The precore gene of the woodchuck hepatitis virus genome is not essential for viral replication in the natural host.

A number of naturally occurring hepatitis B virus mutants that cannot synthesize the virus precore protein have been identified. Such mutants have been associated with more severe forms of hepatitis, including fulminant hepatitis. The most common mutation observed is a substitution of G to A in the distal precore gene that converts a codon specifying Trp (TGG) to a termination codon (TAG). Using oligonucleotide-directed mutagenesis, we have produced the same point mutation in the precore gene of an infectious clone of woodchuck hepatitis virus (WHV). Transfection of mutant WHV DNA into the livers of adult woodchucks resulted in replication of the mutant in three of three susceptible animals. Levels of virus replication and transient elevations in liver enzymes in serum were similar to those of adult animals infected with wild-type WHV. Virions, found to possess mutant precore genes by polymerase chain reaction amplification and DNA sequencing, were recovered from the serum of one of the animals and inoculated subcutaneously into neonatal woodchucks. They produced infection in all five animals studied. The level of virus replication in neonatal animals infected with this mutant virus was comparable to that found in neonatal woodchucks infected with wild-type WHV, but none of five woodchucks infected with the precore mutant virus as neonates became chronic virus carriers. It was concluded that the precore gene of the WHV genome is not essential for virus replication in the natural host but may be important for chronic infection.     

Chemoimmunotherapy of chronic hepatitis B virus infection in the woodchuck model overcomes immunologic tolerance and restores T-cell responses to pre-S and S regions of the viral envelope protein

Treatment of chronic hepatitis B virus (HBV) infection could combine potent antiviral drugs and therapeutic vaccines to overcome immunological tolerance and induce the recovery phenotype to protect against disease progression. Conventional vaccination of woodchucks chronically infected with the woodchuck hepatitis virus (WHV) elicited differential T-cell response profiles depending on whether or not carriers were treated with the potent antiviral drug clevudine (CLV), which significantly reduces viral and antigen loads. The differential T-cell responses defined both CLV-dependent and CLV-independent epitopes of the pre-S and S regions of the WHV envelope protein. Only combined treatment involving CLV and conventional vaccine therapeutically restored the T-cell response profile of chronic WHV carrier woodchucks to that seen in prophylactic vaccination and in recovery from acute WHV infection. The results have implications for mechanisms of immunological tolerance operating in chronic HBV infection and suggest that such combined chemoimmunotherapy may be useful for treatment of humans with chronic HBV infection.     

Combination of DNA Prime – Adenovirus Boost Immunization with Entecavir Elicits Sustained Control of Chronic Hepatitis B in the Woodchuck Model

A potent therapeutic T-cell vaccine may be an alternative treatment of chronic hepatitis B virus (HBV) infection. Previously, we developed a DNA prime-adenovirus (AdV) boost vaccination protocol that could elicit strong and specific CD8⁺ T-cell responses to woodchuck hepatitis virus (WHV) core antigen (WHcAg) in mice. In the present study, we first examined whether this new prime-boost immunization could induce WHcAg-specific T-cell responses and effectively control WHV replication in the WHV-transgenic mouse model. Secondly, we evaluated the therapeutic effect of this new vaccination strategy in chronically WHV-infected woodchucks in combination with a potent antiviral treatment. Immunization of WHV-transgenic mice by DNA prime-AdV boost regimen elicited potent and functional WHcAg-specific CD8⁺ T-cell response that consequently resulted in the reduction of the WHV load below the detection limit in more than 70% of animals. The combination therapy of entecavir (ETV) treatment and DNA prime-AdV boost immunization in chronic WHV carriers resulted in WHsAg- and WHcAg-specific CD4⁺ and CD8⁺ T-cell responses, which were not detectable in ETV-only treated controls. Woodchucks receiving the combination therapy showed a prolonged suppression of WHV replication and lower WHsAg levels compared to controls. Moreover, two of four immunized carriers remained WHV negative after the end of ETV treatment and developed anti-WHs antibodies. These results demonstrate that the combined antiviral and vaccination approach efficiently elicited sustained immunological control of chronic hepadnaviral infection in woodchucks and may be a new promising therapeutic strategy in patients.     

Prime/Boost Immunization with DNA and Adenoviral Vectors Protects from Hepatitis D Virus (HDV) Infection after Simultaneous Infection with HDV and Woodchuck Hepatitis Virus

Hepatitis D virus (HDV) superinfection of hepatitis B virus (HBV) carriers causes severe liver disease and a high rate of chronicity. Therefore, a vaccine protecting HBV carriers from HDV superinfection is needed. To protect from HDV infection an induction of virus-specific T cells is required, as antibodies to the two proteins of HDV, p24 and p27, do not neutralize the HBV-derived envelope of HDV. In mice, HDV-specific CD8⁺ and CD4⁺ T cell responses were induced by a DNA vaccine expressing HDV p27. In subsequent experiments, seven naive woodchucks were immunized with a DNA prime and adenoviral boost regimen prior to simultaneous woodchuck hepatitis virus (WHV) and HDV infection. Five of seven HDV-immunized woodchucks were protected against HDV infection, while acute self-limiting WHV infection occurred as expected. The two animals with the breakthrough had a shorter HDV viremia than the unvaccinated controls. The DNA prime and adenoviral vector boost vaccination protected woodchucks against HDV infection in the setting of simultaneous infection with WHV and HDV. In future experiments, the efficacy of this protocol to protect from HDV infection in the setting of HDV superinfection will need to be proven.     

Enhancing Virus-Specific Immunity In Vivo by Combining Therapeutic Vaccination and PD-L1 Blockade in Chronic Hepadnaviral Infection

Hepatitis B virus (HBV) persistence is facilitated by exhaustion of CD8 T cells that express the inhibitory receptor programmed cell death-1 (PD-1). Improvement of the HBV-specific T cell function has been obtained in vitro by inhibiting the PD-1/PD-ligand 1 (PD-L1) interaction. In this study, we examined whether in vivo blockade of the PD-1 pathway enhances virus-specific T cell immunity and leads to the resolution of chronic hepadnaviral infection in the woodchuck model. The woodchuck PD-1 was first cloned, characterized, and its expression patterns on T cells from woodchucks with acute or chronic woodchuck hepatitis virus (WHV) infection were investigated. Woodchucks chronically infected with WHV received a combination therapy with nucleoside analogue entecavir (ETV), therapeutic DNA vaccination and woodchuck PD-L1 antibody treatment. The gain of T cell function and the suppression of WHV replication by this therapy were evaluated. We could show that PD-1 expression on CD8 T cells was correlated with WHV viral loads during WHV infection. ETV treatment significantly decreased PD-1 expression on CD8 T cells in chronic carriers. In vivo blockade of PD-1/PD-L1 pathway on CD8 T cells, in combination with ETV treatment and DNA vaccination, potently enhanced the function of virus-specific T cells. Moreover, the combination therapy potently suppressed WHV replication, leading to sustained immunological control of viral infection, anti-WHs antibody development and complete viral clearance in some woodchucks. Our results provide a new approach to improve T cell function in chronic hepatitis B infection, which may be used to design new immunotherapeutic strategies in patients.     

Monitoring of early events of experimental woodchuck hepatitis infection: Studies of peripheral blood mononuclear cells by cytofluorometry and PCR

Abstract The peripheral blood mononuclear cells (PBMC) of woodchucks experimentally infected by woodchuck hepatitis virus (WHV) were examined simultaneously for the presence of membrane associated WHV antigens by cytofluorometry, and for WHV DNA and RNA sequences by the polymerase chain reaction (PCR). Four woodchucks were inoculated: two with a well-defined infectious inoculum and two with an inoculum obtained from an animal at the late incubation phase, which was positive for WHV DNA by PCR but still devoid of WHV markers. Infection was demonstrated in all four inoculated woodchucks by the appearance at different times of WHV DNA and WHV antigens in both leucocytes and serum. WHV DNA was first detected by PCR either in the serum (two cases) or in leucocytes (two cases). The mean percentage of cells positive for membrane associated WHsAg or WHcAg detected by cytofluorometry were 37%±25 and 17%±15 respectively. After 8 weeks, all inoculated animals were WHsAg positive in serum. These data suggest that PBMC are involved in the early events of hepadnavirus infection. They also show that sera which are positive by PCR for WHV DNA may transmit viral infection even while still seronegative for WHV markers and for WHV DNA by dot blot.     

Immunization with surface antigen vaccine alone and after treatment with 1-(2-fluoro-5-methyl-beta-L-arabinofuranosyl)-uracil (L-FMAU) breaks humoral and cell-mediated immune tolerance in chronic woodchuck hepatitis virus infection

Woodchucks chronically infected with the woodchuck hepatitis virus (WHV) were treated with the antiviral drug 1-(2-fluoro-5-methyl-beta-L-arabinofuranosyl)-uracil (L-FMAU) or placebo for 32 weeks. Half the woodchucks in each group then received four injections of surface antigen vaccine during the next 16 weeks. Vaccination alone elicited a low-level antibody response to surface antigen in most carriers but did not affect serum WHV DNA and surface antigen. Carriers treated first with L-FMAU to reduce serum WHV DNA and surface antigen and then vaccinated had a similar low-level antibody response to surface antigen. Following vaccinations, cell-mediated immunity to surface antigen was demonstrated in both groups, independent of serum viral and antigen load, but was significantly enhanced in woodchucks treated with L-FMAU and was broadened to include other viral antigens (core, e, and x antigens and selected core peptides). Cell-mediated immunity and antibody responses to surface antigen were observed after drug discontinuation in half of the carriers that received L-FMAU alone. Surface antigen vaccine alone or in combination with drug broke humoral and cell-mediated immune tolerance in chronic WHV infection, but the combination with drug was more effective. This suggested that a high viral and antigen load in carriers is important in maintaining immunologic tolerance during chronicity. The humoral and cellular immunity associated with the combination of L-FMAU and vaccine resembled that observed in self-limited WHV infection. Such combination therapy represents a potentially useful approach to the control of chronic hepatitis B virus infection in humans.     

Cyclosporin A modulates the course of woodchuck hepatitis virus infection and induces chronicity.

Immunosuppression is known to influence the state of chronic hepatitis B virus infection, and is thought to increase the risk of developing chronic infection in newly exposed individuals. Cyclosporin A (CsA), an immunosuppressive agent that inhibits Th cell function, was administered to woodchucks chronically infected with woodchuck hepatitis virus (WHV), and resulted in a decreased severity of chronic hepatitis and an increased viremia during the treatment. Adult woodchucks inoculated with WHV and given CsA for 14 wk had increased viremias, decreased acute phase liver injury, and developed chronic infections at a higher rate compared with immunocompetent woodchucks given virus alone (chronicity in seven of seven WHV + CsA + vs zero of nine WHV + CsA-; p less than 0.001). These results in a relevant animal model of hepatitis B virus infection indicate: 1) that liver injury in acute hepadnavirus infections is immune-mediated and not a direct cytopathic effect of virus replication; 2) that Th cells function in the inflammatory response and in the immunologic control of hepadnavirus infection; and 3) that suppression of Th cell function in acute hepadnavirus infection decreases liver injury but alters the outcome of infection in favor of chronicity. These results also suggest continued challenges in the application of CsA in liver transplantation for hepatitis B virus-induced diseases.     

Hepadnavirus infection of peripheral blood lymphocytes in vivo: woodchuck and chimpanzee models of viral hepatitis.

The peripheral blood lymphocytes (PBL) of five hepatitis B virus (HBV)-infected chimpanzees and 17 woodchuck hepatitis virus (WHV)-infected woodchucks were examined for the presence of viral DNA and RNA. HBV DNA was detected in the PBL of three of three chronically infected chimpanzees but in neither of two animals with acute HBV infection. WHV DNA was found in the PBL of 11 of 13 chronically infected woodchucks and in the PBL and bone marrow of 1 of 4 woodchucks with antibody to WHV surface antigen. Viral DNA in the PBL and bone marrow was episomal, primarily existing as multimers with some monomeric forms. Integrated HBV DNA was detected in the PBL of one chronically infected chimpanzee, but only for a brief period. Viral RNA was also detected in the PBL, although less frequently than was DNA. HBV RNA in chimpanzee PBL existed as 3.8- and 7.5-kilobase species, while 2.3- and 3.8-kilobase WHV RNA was found in woodchuck PBL. Subfractionation of PBL isolated from the chronically infected chimpanzees demonstrated that HBV DNA and RNA were located in B and T cells. No HBV DNA was detected in the macrophages. These results, along with the recent reports of HBV nucleic acids in the PBL of human patients, suggest that infection of PBL may be a general phenomenon associated with the pathology of hepadnaviruses.     

Protection of chimpanzees from type B hepatitis by immunization with woodchuck hepatitis virus surface antigen.

Two chimpanzees immunized with woodchuck hepatitis virus (WHV) surface antigen (WHsAg) developed antibodies cross-reactive with hepatitis B virus (HBV) surface antigen (HBsAg). After challenge with HBV, one animal was completely protected and the other experienced a subclinical infection, without evidence of liver disease. Three woodchucks immunized with HBsAg developed antibodies to HBsAg which did not cross-react with WHsAg. After challenge with WHV, all three woodchucks developed typical acute infections with associated hepatic lesions. Serological studies with the cross-reactive antibodies raised in chimpanzees suggested that the protective epitopes of WHsAg were related to the group a specificity of HBsAg. These studies indicated that cross-protective epitopes are shared by HBV and WHV; however, the humoral response to these epitopes can vary among species.     

Woodchuck hepatitis virus is a more efficient oncogenic agent than ground squirrel hepatitis virus in a common host.

Chronic infection with hepatitis B viruses (hepadnaviruses) is a major cause of hepatocellular carcinoma (HCC), but the incubation time varies from 1 to 2 years to several decades in different host species infected with indigenous viruses. To discern the influence of viral and host factors on the kinetics of induction of HCC, we exploited the recent observation that ground squirrel hepatitis virus (GSHV) is infectious in woodchucks (C. Seeger, P. L. Marion, D. Ganem, and H. E. Varmus, J. Virol. 61:3241-3247, 1987) to compare the pathogenic potential of GSHV and woodchuck hepatitis virus (WHV) in chronically infected woodchucks. Chronic GSHV infection in woodchucks produces mild to moderate portal hepatitis, similar to that observed in woodchucks chronically infected with WHV. However, HCC developed in GSHV carriers about 18 months later than in WHV carriers. Thus, although both viruses are oncogenic in woodchucks, GSHV and WHV differ in oncogenic determinants that can affect the kinetics of appearance of HCC in chronically infected animals.     

Replication of naturally occurring woodchuck hepatitis virus deletion mutants in primary hepatocyte cultures and after transmission to naive woodchucks

Woodchuck hepatitis virus (WHV) mutants with core internal deletions (CID) occur naturally in chronically WHV-infected woodchucks, as do hepatitis B virus mutants in humans. We studied the replication of WHV deletion mutants in primary woodchuck hepatocyte cultures and in vivo after transmission to naive woodchucks. By screening 14 wild-caught, chronically WHV-infected woodchucks, two woodchucks, WH69 and WH70, were found to harbor WHV CID mutants. Consistent with previous results, WHV CID mutants from both animals had deletions of variable lengths (90 to 135 bp) within the middle of the WHV core gene. In woodchuck WH69, WHV CID mutants represented a predominant fraction of the viral population in sera, normal liver tissues, and to a lesser extent, in liver tumor tissues. In primary hepatocytes of WH69, the replication of wild-type WHV and CID mutants was maintained at least for 7 days. Although WHV CID mutants were predominant in fractions of cellular WHV replicative intermediates, mutant covalently closed circular DNAs (cccDNAs) appeared to be a small part of cccDNA-enriched fractions. Analysis of cccDNA-enriched fractions from liver tissues of other woodchucks confirmed that mutant cccDNA represents only a small fraction of the total cccDNA pool. Four naive woodchucks were inoculated with sera from woodchuck WH69 or WH70 containing WHV CID mutants. All four woodchucks developed viremia after 3 to 4 weeks postinoculation (p.i.). They developed anti-WHV core antigen (WHcAg) antibody, lymphoproliferative response to WHcAg, and anti-WHV surface antigen. Only wild-type WHV, but no CID mutant, was found in sera from these woodchucks. The WHV CID mutant was also not identified in liver tissue from one woodchuck sacrificed in week 7 p.i. Three remaining woodchucks cleared WHV. Thus, the presence of WHV CID mutants in the inocula did not significantly change the course of acute self-limiting WHV infection. Our results indicate that the replication of WHV CID mutants might require some specific selective conditions. Further investigations on WHV CID mutants will allow us to have more insight into hepadnavirus replication.     

DNA Prime-Adenovirus Boost Immunization Induces a Vigorous and Multifunctional T-Cell Response against Hepadnaviral Proteins in the Mouse and Woodchuck Model

Induction of hepatitis B virus (HBV)-specific cytotoxic T cells by therapeutic immunization may be a strategy to treat chronic hepatitis B. In the HBV animal model, woodchucks, the application of DNA vaccine expressing woodchuck hepatitis virus (WHV) core antigen (WHcAg) in combination with antivirals led to the prolonged control of viral replication. However, it became clear that the use of more potent vaccines is required to overcome WHV persistence. Therefore, we asked whether stronger and more functional T-cell responses could be achieved using the modified vaccines and an optimized prime-boost vaccination regimen. We developed a new DNA plasmid (pCGWHc) and recombinant adenoviruses (AdVs) showing high expression levels of WHcAg. Mice vaccinated with the improved plasmid pCGWHc elicited a stronger WHcAg-specific CD8⁺ T-cell response than with the previously used vaccines. Using multicolor flow cytometry and an in vivo cytotoxicity assay, we showed that immunization in a DNA prime-AdV boost regimen resulted in an even more vigorous and functional T-cell response than immunization with the new plasmid alone. Immunization of naïve woodchucks with pCGWHc plasmid or AdVs induced a significant WHcAg-specific degranulation response prior to the challenge, this response had not been previously detected. Consistently, this response led to a rapid control of infection after the challenge. Our results demonstrate that high antigen expression levels and the DNA prime-AdV boost immunization improved the T-cell response in mice and induced significant T-cell responses in woodchucks. Therefore, this new vaccination strategy may be a candidate for a therapeutic vaccine against chronic HBV infection.     

Failure to detect polyalbumin-binding sites on the woodchuck hepatitis virus surface antigen: implications for the pathogenesis of hepatitis B virus in humans.

Binding sites for polymerized albumin on hepatitis B virus components were reported in human hepatitis B virus chronic carriers predominantly with active viral replication (HB e antigen positive). The presence of comparable albumin-binding sites in the woodchuck hepatitis virus (WHV) model was examined on WHV components obtained from woodchucks with active viral replication (DNA polymerase positive). Binding sites for polymerized woodchuck serum albumin were not detected on the intact WHV virion, on 22-nm woodchuck hepatitis surface antigen (WHsAg), or on WHsAg polypeptides. Woodchuck albumin was not detected in purified 22-nm WHsAg, and anti-albumin antibodies were not detected in WHV chronic-carrier woodchucks. Our results in the WHV model argue against a role for viral polyalbumin-binding sites in tissue- and host-specific virus infectivity.     

Characterization of T-cell response to woodchuck hepatitis virus core protein and protection of woodchucks from infection by immunization with peptides containing a T-cell epitope.

Specific activation of T cells appears to be a prerequisite for viral clearance during hepatitis B virus (HBV) infection. The T-cell response to HBV core protein is essential in determining an acute or chronic outcome of HBV infection, but how this immune response contributes to the course of infection remains unclear. This is due to results obtained from humans, which are restricted to phenomenological observations occurring during the clinical onset after HBV infection. Thus, a useful animal model is needed. Characterization of the T-cell response to the core protein (WHcAg) of woodchuck hepatitis virus (WHV) in woodchucks contributes to the understanding of these mechanisms. Therefore, we investigated the response of woodchuck peripheral blood mononuclear cells (PBMCs) to WHcAg and WHcAg-derived peptides, using our 5-bromo-2'-deoxyuridine assay. We demonstrated WHcAg-specific proliferation of PBMCs and nylon wool-nonadherent cells from acutely WHV-infected woodchucks. Using a cross-reacting anti-human T-cell (CD3) antiserum, we identified nonadherent cells as woodchuck T cells. T-cell epitope mapping with overlapping peptides, covering the entire WHcAg, revealed T-cell responses of acutely WHV-infected woodchucks to peptide1-20, peptide100-119, and peptide112-131. Detailed epitope analysis in the WHcAg region from amino acids 97 to 140 showed that T cells especially recognized peptide97-110. Establishment of polyclonal T-cell lines with WHcAg or peptide97-110 revealed reciprocal stimulation by peptide97-110 or WHcAg, respectively. We vaccinated woodchucks with peptide97-110 or WHcAg to prove the importance of this immunodominant T-cell epitope. All woodchucks immunized with peptide97-110 or WHcAg were protected. Our results show that the cellular immune response to WHcAg or to one T-cell epitope protects woodchucks from WHV infection.     

Treatment of Chronic Viral Hepatitis in Woodchucks by Prolonged Intrahepatic Expression of Interleukin-12

Chronic hepatitis B is a major cause of liver-related death worldwide. Interleukin-12 (IL-12) induction accompanies viral clearance in chronic hepatitis B virus infection. Here, we tested the therapeutic potential of IL-12 gene therapy in woodchucks chronically infected with woodchuck hepatitis virus (WHV), an infection that closely resembles chronic hepatitis B. The woodchucks were treated by intrahepatic injection of a helper-dependent adenoviral vector encoding IL-12 under the control of a liver-specific RU486-responsive promoter. All woodchucks with viral loads below 10¹⁰ viral genomes (vg)/ml showed a marked and sustained reduction of viremia that was accompanied by a reduction in hepatic WHV DNA, a loss of e antigen and surface antigen, and improved liver histology. In contrast, none of the woodchucks with higher viremia levels responded to therapy. The antiviral effect was associated with the induction of T-cell immunity against viral antigens and a reduction of hepatic expression of Foxp3 in the responsive animals. Studies were performed in vitro to elucidate the resistance to therapy in highly viremic woodchucks. These studies showed that lymphocytes from healthy woodchucks or from animals with low viremia levels produced gamma interferon (IFN-γ) upon IL-12 stimulation, while lymphocytes from woodchucks with high viremia failed to upregulate IFN-γ in response to IL-12. In conclusion, IL-12-based gene therapy is an efficient approach to treat chronic hepadnavirus infection in woodchucks with viral loads below 10¹⁰ vg/ml. Interestingly, this therapy is able to break immunological tolerance to viral antigens in chronic WHV carriers.Hepatitis B virus (HBV) infection is estimated to cause approximately 1 million deaths per year (http://www.who.int/emc). Current therapies against chronic HBV include pegylated alpha interferon (IFN-α) and nucleoside/nucleotide analogs, such as lamivudine, entecavir, adefovir, and tenofovir (16). Sustained antiviral responses are achieved in only one-third of the patients treated with pegylated IFN-α (16, 32). Nucleoside/nucleotide analogs are effective, but treatment must be continued for many years, resulting in high costs, the emergence of drug-resistant variants, and frequent relapses after the discontinuation of therapy (32).Chronic HBV infection is associated with defects in antiviral immunity (3). Patients with an acute self-limiting HBV infection develop neutralizing anti-HBs antibodies and multispecific CD4⁺ and CD8⁺ T-cell responses with a type 1 cytokine profile (3). In contrast, patients with chronic HBV infection show no protective humoral immunity and a weak or undetectable virus-specific T-cell response (5). The precise mechanism responsible for this immunotolerance is still unknown. Recently, several studies have indicated that regulatory T cells (Tregs), immunosuppressive cytokines, and inhibitory receptor-ligand interactions, such as PD1-PDL1, contribute to the impairment of virus-specific T-cell responses in chronic HBV infection (1, 17, 23, 26, 28).Interleukin-12 (IL-12) is a cytokine produced by antigen-presenting cells that is essential for the induction of effective cell-mediated immunity against viruses and other pathogens (30). IL-12 promotes Th1-type responses, enhances cytotoxic-T-cell activity, and stimulates T lymphocytes and NK cells to produce IFN-γ (30). The administration of recombinant IL-12 (rIL-12) to HBV-transgenic mice resulted in the inhibition of HBV replication in the liver (8). In vitro studies demonstrated that IL-12 was able to enhance HBV-specific T-cell responses in chronic HBV carriers (15, 22, 31). Moreover, the upregulation of IL-12 production has been shown to be associated with HBe seroconversion, indicating an important role for this cytokine in the control of HBV infection (22). In two studies, rIL-12 administered to patients with chronic HBV infection once per week as monotherapy (7) or twice weekly in combination with lamivudine (21) increased virus-specific T-cell reactivity and exerted significant antiviral activity. However, in both studies, a rebound of viremia occurred following drug withdrawal. The antiviral effects of rIL-12 were dose dependent, but the therapy was limited by severe toxicity when high doses of the cytokine were used (7, 21).Gene therapy can significantly increase cytokine expression in the target organ without excessively elevating systemic cytokine levels, which leads to an increased efficacy/toxicity ratio. In the present study, we tested the antiviral potential of IL-12-mediated gene therapy using a high-capacity adenovirus (HC-Ad) encoding murine IL-12 (mIL-12) under the control of a liver-specific inducible promoter that is responsive to the progesterone antagonist RU486 (30). HC-Ad is a nonintegrating vector characterized by strong hepatotropism, low toxicity, long-term transgene expression, and high cloning capacity (13). All these properties make HC-Ad a useful tool for therapeutic applications in human liver diseases.As an animal model of chronic HBV infection, we used woodchucks that were chronically infected with woodchuck hepatitis virus (WHV). WHV is a hepadnavirus with a genomic organization, biological properties, and a replicative strategy that are essentially identical to those of HBV. When WHV infects woodchucks in the perinatal period of life, the infection causes chronic hepatitis in most animals. This condition resembles the pathological features and natural history of chronic hepatitis B (18). Here, we demonstrate that prolonged intrahepatic expression of IL-12 overcomes immunological tolerance for WHV antigens and induces sustained antiviral effects in woodchucks with chronic WHV infection and viral loads below 10¹⁰ viral genomes (vg)/ml. These observations indicate that IL-12 gene therapy is an alternative approach for the treatment of chronic HBV infection.     

Combination therapy with lamivudine and adenovirus causes transient suppression of chronic woodchuck hepatitis virus infections

Treatment of hepatitis B virus carriers with the nucleoside analog lamivudine suppresses virus replication. However, rather than completely eliminating the virus, long-term treatment often ends in the outgrowth of drug-resistant variants. Using woodchucks chronically infected with woodchuck hepatitis virus (WHV), we investigated the consequences of combining lamivudine treatment with immunotherapy mediated by an adenovirus superinfection. Eight infected woodchucks were treated with lamivudine and four were infected with approximately 10(13) particles of an adenovirus type 5 vector expressing beta-galactosidase. Serum samples and liver biopsies collected following the combination therapy revealed a 10- to 20-fold reduction in DNA replication intermediates in three of four woodchucks at 2 weeks after adenovirus infection. At the same time, covalently closed circular DNA (cccDNA) and viral mRNA levels both declined about two- to threefold in those woodchucks, while mRNA levels for gamma interferon and tumor necrosis factor alpha as well as for the T-cell markers CD4 and CD8 were elevated about twofold. Recovery from adenovirus infection was marked by elevation of sorbitol dehydrogenase, a marker for hepatocyte necrosis, as well as an 8- to 10-fold increase in expression of proliferating cell nuclear antigen, a marker for DNA synthesis, indicating significant hepatocyte turnover. The fact that replicative DNA levels declined more than cccDNA and mRNA levels following adenovirus infection suggests that the former decline either was cytokine induced or reflects instability of replicative DNA in regenerating hepatocytes. Virus titers in all four woodchucks were only transiently suppressed, suggesting that the effect of combination therapy is transient and, at least under the conditions used, does not cure chronic WHV infections.     

Natural history of experimental woodchuck hepatitis virus infection: molecular virologic features of the pancreas, kidney, ovary, and testis.

The kinetic patterns of woodchuck hepatitis virus (WHV) infection were monitored in the pancreas, kidneys, ovaries, and testes. Groups of woodchucks experimentally infected with a standardized inoculum of WHV were sacrificed at different times over a 65-week period beginning in the preacute phase of viral infection and continuing to the period of serologic recovery or the establishment of chronic infections and subsequent hepatocellular carcinoma (B. E. Korba, P. J. Cote, F. V. Wells, B. Baldwin, H. Popper, R. H. Purcell, B. C. Tennant, and J. L. Gerin, J. Virol. 63:1360-1370, 1989). Tissues from an additional group of long-term (2 to 3 years) chronic WHV carriers which had been infected with the same WHV inocula were also examined. Viral DNA replication intermediates were found in all four tissues during the acute phase of WHV infection. However, WHV DNA replication intermediates were observed only in the kidneys of a small proportion of the chronically infected animals. Following the acute phase of infection, WHV DNA was present only in the pancreas, kidneys, and ovaries of the chronically infected woodchucks. A progressive evolution of different WHV genomic forms related to the replicative state of WHV was observed in these tissues. Histologic evaluation of these four tissues revealed only minimal, localized lesions which were not correlated with the state of WHV activity. The observations compiled in this study further extend the tissue tropism of WHV.     

Protection against woodchuck hepatitis virus (WHV) infection by gene gun coimmunization with WHV core and interleukin-12

Woodchuck hepatitis virus (WHV) and hepatitis B virus (HBV) are closely similar with respect to genomic organization, host antiviral responses, and pathobiology of the infection. T-cell immunity against viral nucleocapsid (HBcAg or WHcAg) has been shown to play a critical role in viral clearance and protection against infection. Here we show that vaccination of healthy woodchucks by gene gun bombardment with a plasmid coding for WHcAg (pCw) stimulates proliferation of WHcAg-specific T cells but that these cells do not produce significant levels of gamma interferon (IFN-gamma) upon antigen stimulation. In addition, animals vaccinated with pCw alone were not protected against WHV inoculation. In order to induce a Th1 cytokine response, another group of woodchucks was immunized with pCw together with another plasmid coding for woodchuck interleukin-12 (IL-12). These animals exhibited WHcAg-specific T-cell proliferation with high IFN-gamma production and were protected against challenge with WHV, showing no viremia or low-level transient viremia after WHV inoculation. In conclusion, gene gun immunization with WHV core generates a non-Th1 type of response which does not protect against experimental infection. However, steering the immune response to a Th1 cytokine profile by IL-12 coadministration achieves protective immunity. These data demonstrate a crucial role of Th1 responses in the control of hepadnavirus replication and suggest new approaches to inducing protection against HBV infection.